This is a revised application including the addition of two new specific aims, which outlines research intended to (a) further test the hypothesis that the current national and international estimates of the minimum physiological requirements for the nutritionally Indispensable amino acids in healthy adults are far too low and (b) strengthen and validate our tentative new estimates of the Indispensable amino acid requirements in adults, which could become the standard reference values for practical use in adult human protein and amino acid nutrition. With leucine as the test amino acid we have previously conducted 24h Isotope tracer infusion studies to validate the concept of whole-body tracer balance. Because the 24h protocol current serves as a "gold standard", we now propose to extend this approach to investigations of the aromatic amino acids (phenylalanine/tyrosine) lysine and threonine because the metabolic characteristics of all of these amino acids are different from leucine (Specific Aim 1). The 24h infusion studies in adult men and women will be conducted following adjustment to graded intakes of these indispensable amino acids. Additionally we will explore (Specific Aim 2) the effect of mode of feeding and route of 13C-tracer administration on the oxidative loss of specific indispensable amino acids. In Specific Aim 3 we will assess the physiological significance of the urea-N salvage pathway, principally under the experimental conditions we have used to study amino acid kinetics and amino acid requirements. Finally, (Specific Aim 4) the predictive method for estimating amino acid requirements, based on measurement of obligatory oxidative losses, will be validated in a series of diet-tracer studies with leucine, lysine, phenylalanine and methionine as test amino acids. The objective of this integrated series of studies is to further understand why current, internationally accepted amino acid requirement values differ so markedly from those based on tracer techniques and to strengthen and improve upon our tentative, new requirement values. Additionally, these studies will generate meaningful new data on the quantitative aspects of the physiology of human amino acid metabolism, which potentially will lead to better methods and diagnostic tools for evaluating nutritional status and requirements.